This invention relates generally to the analysis and characterization of biomolecules, complexes comprising biomolecules or analogous species thereof. The results of the analysis, represented as signatures, can be used to establish relationships between properties of large numbers of species which allows selection of species for specific uses based upon the correlation of the species' properties which have been analyzed and characterized using the methods of the invention.
Biomolecules are generally flexible three-dimensional (3-D) molecular structures. Members of one prototypical class of biomolecules, polypeptides, are normally constructed of a linear arrangement amino acids, i.e., they comprise amino acid sequences. The majority of amino acids which are contained in polypeptides are the 20 commonly-known amino acids. With proteins, as well as other biomolecules, the particular details of the 3-D structure of the protein is known to determine much of the biological information specific to a specific biomolecule. Typically, changes in the 3-D structure (conformation) are associated with a specific biological effect. In this sense, the state of conformation of a biomolecule must therefore reflect certain aspects of the biological information specific to the biomolecule.
It is generally recognized that the biological information of a specific biomolecule can be transferred to or from another species by non-covalent interactions between the specific biomolecule and the other species. Examples of such non-covalent interaction include binding events wherein the binding of a smaller molecule to the biomolecule induces changes in the conformation of the biomolecule. In many cases, as will be recognized by those of skill in the art, these changes in the conformation of the biomolecule can result in further changes in either the behavior or the characteristics of the biomolecules involved. These changes are often quite significant and include changes in what other biomolecules can be bound by the biomolecule. Series of events such as these are at the root of many specific biological effects. Therefore, analysis of biomolecular structures is of significant interest in the pursuit of effective therapies and improvements to biotechnology.
As it is generally practiced, structural analysis of biomolecules involves examination of structure at a number of levels of detail. For proteins, this includes analysis at the level of amino acid sequence (primary structure), secondary structure (e.g., alpha helix or beta sheet composition), tertiary structure (3-D detailed atomic structure), and quaternary structure (when the biomolecule is comprised of discrete sub-domains). Techniques to evaluate each of these aspects of structure are generally specific to the type of information desired. For example, the preferred methods for determining the primary sequence of a biomolecule are usually incapable of providing tertiary structural information and the techniques for providing tertiary structural information are ill-suited for determining primary sequence. Primarily, the methods and instrumentation that are available for analyzing higher order structure, namely, conformational information, are complex, slow, and difficult to perform. This is not surprising since detailed 3-D information involves, by definition, resolution of the type of atoms and their coordinates in space at the atomic scale. A different approach is needed to rapidly obtain useful higher order structural information with ease and simplicity.
Many biological processes are mediated by noncovalent binding interactions between a protein and another molecule. Examples of these include the participation of receptors, such as those for hormones, messengers and/or drugs, and the binding partners for the receptors, such as hormones, messengers, drugs and/or any other ligand. Such interactions and their role in mediating biological processes are well-known to those of skill in the art. Further, biological processes involving the interaction of one biomolecule with another are commonly recognized to include the interactions between different proteins. It is also recognized that the identification and characterization of interactions such as those described above is of great significance in the process of drug discovery and development. It is also recognized that methods for analyzing biochemical binding interactions, particularly for purposes of gaining insight regarding structural characteristics, require too much time and effort to be generally applied to larger numbers of potentially relevant or useful binding pairs.
Binding of a given receptor to its binding partner may be detected by a variety of techniques capable of monitoring changes in the physico-chemical features of the receptor induced by the binding or by those sensitive to the concentration of the unbound binding partner. The techniques based on the competition replacement of a specific ligand bound to the receptor by a binding partner are particularly popular in the art. Often, the specific ligand of interest is fluorescently or radioactively labeled. When this is done, the appearance of the ligand in a free unbound form resulting from its replacement in the complex with the receptor by a binding partner is readily monitored. However, these techniques are hampered by the fact that only compounds with the affinity for the receptor exceeding that of the specific ligand are capable of replacing the ligand in significant quantities and thereby allow its detection. Further, there are significant waste handling problems associated with the methods used to render a ligand detectable, i.e., radioactivity, toxicity.
In some cases, binding of a ligand is sufficient to change the function of a receptor. If so, then determination of ligand binding can also be used to determine that the function of the receptor has also changed, e.g., altered enzymatic activity or altered affinity for other biomolecules. In these cases, the quantification of ligand-receptor binding is suitable for screening compounds capable of affecting the receptor function and/or activity. However, such a simple approach is not always suitable or useful.
In many cases, binding of a particular ligand results in a specific alteration of the receptor activity and/or function, while binding of another ligand results in a different specific alteration of activity and/or function. These differences in the response to ligand binding depend upon the particular details of changes in the 3-D structure of the receptor (conformational changes). One example of this is the effect of different estrogenic compounds on the estrogen receptor. In this case, different compounds result in different, distinct conformational changes and these different changes result in different activity and/or function of the estrogen receptor. Thus, when screening a class of compounds for their effect on this receptor, or receptors with similar properties, any measure of affinity of ligand for receptor would not provide adequate information in regards to potential pharmacological activity of any compound. Further, the strength of the affinity is, in general, not correlated with the specific details of the conformational changes in the receptor. This lack of correlation between affinity and structure renders any predictive effort based on simple affinity characterization even less likely to succeed. However, the alternative, gaining conformational information that may be useful, is very difficult and expensive.
The experimental work required to characterize the state of conformation of a biomolecule, or of a complex containing a biomolecule such as a receptor-ligand complex, is extensive. Information about specific conformational changes may be obtained by indirect methods such as, but not limited to, spectroscopic, hydrodynamic, or immunogenic characterization of the receptor molecule. Alternatively, more direct methods such as, but not limited to, crystallization of the biomolecule or co-crystallization of the ligand-receptor complex followed by X-ray diffraction studies and NMR spectroscopy of the biomolecule or the ligand-receptor complex in solution, can be used.
The indirect methods generally detect a single property of the receptor molecule that may or may not be affected by the occurring conformational changes in any particular case. If a change is detected, it is often not particularly useful in monitoring what change in the conformational state has occurred, as it is only one-dimensional. Accordingly, for more complex molecules, the use of indirect techniques is often limited to monitoring systems that are already particularly well characterized.
The direct methods, particularly X-ray and NMR analysis, are the most definitive techniques for characterization of the 3-D structure of a biomolecule or of a receptor-ligand complex. But, like the indirect methods, the direct methods too have major limitations. Particularly, the direct methods require considerable amounts of time, are often unsuitable for studying large proteins (NMR), and require materials that are difficult to obtain, such as crystals of diffraction quality (X-Ray crystallography).
The biological effect of a specific ligand binding to a receptor can be determined by directly monitoring the cellular or physiological effects. While this will normally be required at some point during the development of any drug or pharmaceutical, studying biological effects directly is not a feasible means to screen the great many candidate drugs or potential targets that are currently available, or soon will be available. Simply stated, cell culture and animal trials are vastly too expensive in both time and resources to be applied to the large numbers of potential candidate compounds.
While many useful techniques for characterizing molecules exist, a need exists in the art for additional versatile, simple, powerful techniques for characterizing species (including chemical, biological, or biochemical species), characterizing structural aspects of species, characterizing interaction between various species, and the like.